Smooth muscle is the contractile element in most hollow organs and is essential for many physiological functions including those of vascular tissue, airways, and the gastrointestinal tract. A major regulatory mechanism in controlling contractile activity of smooth muscle is phosphorylation of myosin. The level of myosin phosphorylation reflects the activites of two key enzymes: myosin light chain kinase and myosin phosphatase (MP). Recently it was shown that under some conditions, e.g. on stimulation by several physiological agonists, that MP activity is inhibited and this results in a marked increase of myosin phosphorylation at sub-maximal CA2+ concentrations. The goal of this application is to establish a molecular basis for this novel regulatory mechanism. The MP consists of 3 subunits: a catalytic subunit and two putative regulatory subunits of 130 kD and 20 kD. An important additional finding is that phosphylation of the 130 kD subunits inhibits MP activity. However, the kinase is not identified. The specific aims are designed to investigate three aspects of MP function and its regulation. The first will investigate interactions between different subunits and establish important binding sites, or domains, upon which a molecular framework of the MP holoenzyme can be constructed. The role of the 20 kD subunit is not known. Several techniques will be used including the yeast two-hybrid system, construction and expression of several mutants and various assays of phosphatase activity and protein interactions. The second aim will examine the effects of phosphorylation of the 130 kD subunit. The critical site(s) of phosphorylation will be identified. Based on the plan established for the MP holoenzyme, those interactions that change following phosphorylation and that are involved in the mechanism of inhibition will be identified. The final aim is to characterized the kinase involved and procedures are outlined to establish its identity and also to screen for other substrates that might have a physiological role. A candidate for the 130 kD kinase is a rho-activated kinase and this will be investigated. Ultimately, the action(s) of this kinase must be integrated with the signal transduction pathway(s) in smooth muscle that are initiated at the cell membrane and target the contractile apparatus.